Relationships among superantigen toxin gene profiles,genotypes, and pathogenic characteristics of Staphylococcus aureus isolates from bovine mastitis

Staphylococcus aureus is one of the major etiological agents of bovine mastitis, harboring a wide variety of staphylococcal superantigen (SAg) toxin genes. The SAg toxin genes are reported to be closely associated with the pathogenicity of the Staph. aureus causing the bovine mastitis. This study was conducted to investigate SAg toxin gene profiles and to assess the relationships among SAg toxin genes, genotypes of Staph. aureus, and their pathogenic properties. A total of 327 quarter milk samples were collected from bovine mastitis cases for isolation and identification of pathogens. In total, 35 isolates were identified as Staph. aureus, and the prevalence of Staph. aureus in milk samples was 13.6% (35/256). Polymerase chain reaction (PCR) and randomly amplified polymorphic DNA (RAPD) assays were used to detect the SAg toxin genes and to genotype Staph. aureus strains isolated from milk samples of bovine mastitis in 10 dairy herds located in Ningxia, China, respectively. The results showed that among the Staph. aureus isolates (n = 35), 71.4% (n = 25) of isolates carried at least one SAg toxin gene. In total, 18 SAg genes and 21 different gene combination patterns were detected among these isolates. The most common SAg genes in Staph. aureus isolates were sei, sen, and seu (44.0% each), followed by seo, tst, and etB (28.0% each), etA (24.0%), sem and sep (16.0% each), seb, sec, sed, and sek (12.0% each), and sea and seh genes (8.0% each); the seg, sej, and ser genes were present in 4.0% of the isolates. Three gene combinations were found to be related to mobile genetic elements that carried 2 or more genes. The egc-cluster of the seg-sei-sem-sen-seo genes, located on the pathogenicity island Type I υSaβ, was detected in 16% of isolates. Interestingly, we observed 6 RAPD genotypes (I to VI) in Staph. aureus isolates, and 2 of these genotypes were strongly associated with the severity of bovine mastitis; there was a close relationship between the RAPD genotypes and SAg genes. Isolates of RAPD type III were more frequently associated with clinical and subclinical mastitis, whereas strains of type VI were mostly related to subclinical mastitis. In addition, SAg genes were related to severity of bovine mastitis. We conclude that an obvious relationship exists among RAPD genotypes, SAg toxin genes, and severity of bovine mastitis.     

Distribution of newly described enterotoxin-like genes in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> isolated from ready-to-eat foods in Korea

To investigate the distribution of staphylococcal enterotoxin-like (SEl) genes in Staphylococcus aureus from food, a total of 154 S. aureus isolates from ready-to-eat (RTE) foods in Korea were analyzed by mutiplex PCR for the detection of the following 9 staphylococcal enterotoxin-like genes; sek, sel, sem, sen, seo, sep, seq, ser, and seu. Seventy-nine isolates (51.3%) were found to have at least one of SEl genes. The major SEl genes were sek, sem, sen, and seq. Other SEl genes found in the isolates were seo (21 isolates, 13.6%), seu (12 isolates, 7.8%), sep (8 isolates, 5.2%), sel (7 isolates, 4.5%), and ser (2 isolates, 1.3%). Most (95%) of the isolates with staphylococcal enterotoxin (SE) genes were also carried SEl genes. The genes seg, sei, sem, and sen were all detected in the same isolates. Ninety-seven % of isolates with seg+sei+sem+sen also contained seo or seu. Ninety-four % of isolates with sea+seh were found to coexist with sek+seq. The toxic shock syndrome toxin gene, tst-1, was found in all isolates with egc-2, including seg, sei, sem, sen, and seu. The coexistence of SE and SEl genes in S. aureus isolates from RTE foods can be explained by the mobile genetic elements. Because of the mobile genetic element, SE and SEl genes of S. aureus in foods may be transferable to nontoxigenic S. aureus and other food pathogens. Additional studies must be conducted to prevent spread of pathogenic genes such as enterotoxin gene.     

Enterotoxin genes,enterotoxin production,and methicillin resistance in Staphylococcus aureus isolated from milk and dairy products in Central Italy

A total of 227 Staphylococcus aureus colonies, isolated from 54 samples of raw milk and dairy products of bovine, ovine, caprine and bubaline origin were tested for the presence of genes coding for staphylococcal enterotoxins (SEs/SEls) and for methicillin resistance. Ninety-three colonies, from 31 of the 54 samples (57.4%) and from 18 (69.2%) of the 26 farms of origin tested positive for SEs/SEls genes. Most isolates harboured more than one toxin gene and 15 different toxinotypes were recorded. The most frequent were “sec” gene alone (28.6%), “sea, sed, ser, selj” (20%), “seg, sei” and “seh” (8.6%). The 77 colonies harbouring “classical enterotoxins” genes (sea-sed) were further tested for enterotoxin production, which was assessed for 59.2% of the colonies. Three methicillin-resistant S. aureus (MRSA) isolates were detected in three different ovine milk/dairy product samples (1.3%). All isolates belonged to spa type t127, sequence type 1, clonal complex 1, SCCmec type IVa.     

Emergence of methicillin-resistant Staphylococcus aureus (MRSA) ST8 in raw milk and traditional dairy products in the Tizi Ouzou area of Algeria

Staphylococcus aureus is one of the leading causes of food-borne illness worldwide. Raw milk and dairy products are often contaminated with enterotoxigenic strains of this bacterium. Some of these strains carry antimicrobial resistance, leading to a potential risk for consumers. The aim of this study was to characterize S. aureus strains circulating in raw milk and traditional dairy products for carriage of staphylococcal enterotoxin (se) genes and antimicrobial resistance. Overall, 62 out of 270 samples (23%) were contaminated with S. aureus, and 69 S. aureus strains were identified. We studied the enterotoxin genes using 2 multiplex PCR targeting 11 se genes. Seventeen (24.6%) isolates carried one or more genes encoding for staphylococcal enterotoxins. The most commonly detected se genes were seb and sep, followed by seh, sea, and see. Using the disk diffusion method, we found that resistance to penicillin G and tetracycline was the most common. Eleven isolates of methicillin-resistant S. aureus (MRSA) carried the mecA gene. All MRSA isolates belonged to the same spa type (t024) and sequence type (ST8), and carried the seb and sep enterotoxin genes. However, none of them carried the Panton Valentine leukocidin gene (lukF/S-PV). The presence of enterotoxigenic S. aureus strains, including MRSA, in raw milk and dairy products, raises a serious public health concern, because these strains may cause food poisoning outbreaks, be disseminated to the population, or both.     

The prevalence,genetic diversity and antibiotic resistance of Staphylococcus aureus in milk,whey, and cheese from artisan farm dairies

In this study, coagulase positive staphylococci were detected in 45% of the 69 bovine milk, whey and cheese samples taken from five farm dairies, and all raw milk samples were contaminated. Genetic diversity, staphylococcal enterotoxin genes and antimicrobial susceptibility in putative Staphylococcus aureus isolates were investigated. Sixty-one percent of the 72 isolates analysed belonged to the same pulsed field gel electrophoresis group. The spa-typing revealed seven different spa types, t2678 being the most prevalent, but t127 and t197 were also detected. Sixteen different toxin gene profiles were identified in 87.5% of the isolates with sec and tst being the most frequent (52.5%), followed by seg and seh. All isolates were methicillin-sensitive S. aureus, and sensitive to the 12 antibiotics tested. The prevalence of S. aureus and the high diversity of isolates carrying enterotoxin genes constitute grounds for food safety concern in artisanal cheese making, whether pasteurised or not.     

Enterotoxin-producing Staphylococcus aureus genotype B as a major contaminant in Swiss raw milk cheese

The objective of this study was to characterize Staphylococcus aureus isolates from Swiss raw milk cheeses that had been found to be contaminated with coagulase-positive staphylococci and to estimate the frequency of the various genotypes, in particular the mastitis-associated Staph. aureus genotype B (GTB). The isolates were also tested for staphylococcal enterotoxin (SE) genes and other virulence factors. From 623 coagulase-positive staphylococci isolated from 78 contaminated raw milk cheeses, 609 were found to be Staphylococcus aureus. Genotyping of all Staph. aureus isolates was performed by PCR amplification of the 16S–23S rRNA intergenic spacer region, as this method was used previously to differentiate between mastitis subtypes associated with their clinical outcome. In total, 20 different genotypes were obtained and the 5 most frequently occurring genotypes were distributed in 6.4% or more of the samples. The enterotoxin-producing Staph. aureus GTB, known for its high contagiousness and increased pathogenicity in Swiss mastitis herds, was found to be the most abundant subtype at the sample level (71.8%) as well as among the isolates (62.0%). A subset of 107 isolates of the different genotypes were analyzed for the presence of SE genes and revealed 9 different SE gene patterns, with sed being most frequently detected and 26% being PCR-negative for SE genes. Almost all isolates of the major contaminant GTB contained the SE gene pattern sed, sej, ser, with half of them additionally carrying sea. Production of SE in vitro was consistent with the SE genes detected in most of the cases; however, some isolated GTB did not produce SEA. Staphylococcus aureus Protein A (spa) typing revealed 30 different subtypes and most GTB isolates belonged to the bovine spa type t2953; GTB/t2953 was linked among other subtypes to SE production in cheese and staphylococcal intoxication cases. Furthermore, 1 of the 623 isolates was a methicillin-resistant Staph. aureus, which was an seh-carrying Staph. aureus spa type tbl 0635 (non-GTB). We conclude that control and reduction of enterotoxigenic Staph. aureus GTB in dairy herds in Switzerland will not only prevent economic losses at the farm level but also improve the safety of raw milk cheeses; distribution of methicillin-resistant Staph. aureus via raw milk cheese is of less concern.     

Characterization for enterotoxin production,virulence factors,and antibiotic susceptibility of Staphylococcus aureus isolates from various foods in Portugal

Staphylococcus aureus represents a public health challenge worldwide. The aim of this study was the characterization of different food isolates of S. aureus on the basis of their production of enterotoxins, hemolysins and resistance to antibiotics. A total of 148 coagulase-positive staphylococcal strains isolated from different food origins were identified to the species level. By multiplex PCR, 69% of the isolates were shown to be enterotoxigenic (SEs); the most common were sea seg, sea seg sei and seg sei. According to CLSI [CLSI, Clinical and Laboratory Standards Institute, 2007. Performance Standards for Antimicrobial Susceptibility Testing; Fifteenth Informational Supplement. CLSI document M100-S15. Clinical and Laboratory Standards Institute, Wayne, PA], 38% of the isolates were resistant to oxacillin (≥6 μg/mL; MRSA positives) but only 0.68% showed the presence of mecA gene. 70 and 73% of the S. aureus strains were resistant to β-lactams, ampicillin and penicillin, respectively. The virulence pattern was demonstrated to be origin and strain dependent. These findings emphasise the need to prevent the presence of S. aureus strains and SEs production in foods.     

Determination of Enterotoxigenic and Methicillin Resistant Staphylococcus aureus in Ice Cream

The aim of this study was to determine the prevalence of enterotoxigenic and methicillin‐resistant Staphylococcus aureus in ice creams. After culture‐based identification of isolates, the presence of 16S rRNA and nuc was confirmed by mPCR. S. aureus was identified in 18 of 56 fruity (32.1%), 4 of 32 vanilla (12.5%), and 1 of 12 chocolate (8.3%) ice creams. S. aureus was identified as 38 isolates in 23 ice cream samples by culture‐based techniques, but only 35 isolates were confirmed by PCR as S. aureus. To determine the enterotoxigenic properties of PCR‐confirmed S. aureus isolates, a toxin detection kit was used (SET RPLA®). Of the 12 enterotoxigenic S. aureus isolates, 9 SEB (75%), 1 SED (8.3%), 1 SEB+SED (8.3%), and 1 SEA+SEB+SED (8.3%) expressing isolates were found. The presence of enterotoxin genes (sea, seb, sed) was identified in 13 (37.1%) out of 35 isolates by the mPCR technique. In the ice cream isolates, the sea, seb, and sed genes were detected: 1 sea (7.6%), 9 seb (69.2%), 1 sed (7.6%), 1 seb+sed (7.6%), and 1 sea+seb+sed (7.6%), respectively. The sec gene was not detected in any of these isolates. One of the 35 (2.8%) S. aureus strain was mecA positive.     

Occurrence of foodborne pathogens and characterization of Staphylococcus aureus in cheese produced on farm-dairies

The objective of this study was to address knowledge gaps identified in an earlier risk assessment of Staphylococcus aureus and raw milk cheese. A survey of fresh and short-time ripened cheeses produced on farm-dairies in Sweden was conducted to investigate the occurrence and levels of S. aureus, Listeria monocytogenes and Escherichia coli, to characterize S. aureus isolates with special emphasis on enterotoxin genes, antibiotic resistance, bio-typing and genetic variation, and to collect information related to production practices. In general, the hygienic quality of farm-dairy cheeses appeared to be of an acceptable microbiological quality, e.g. L. monocytogenes and staphylococcal enterotoxin were not detected in cheese samples. However, E. coli and enterotoxigenic S. aureus were frequently found in raw milk cheeses and sometimes at levels that are of concern, especially in fresh cheese. Interestingly, levels in raw milk fresh cheese were significantly lower when starter cultures were used. Up to five S. aureus colonies per cheese, if possible, were characterized and about 70% of isolates carried one or more enterotoxin genes, most common were sec and sea. The Ovine biotype (73%) was most common among isolates from goat milk cheese and the Human biotype (60%) from cow milk cheese. Of all isolates, 39% showed decreased susceptibility to penicillin, but the proportion of isolates from cows' cheese (66%) compared to isolates from goats' cheese (27%) was significantly higher. S. aureus isolates with different properties were detected in cheese from the same farm and, sometimes even the same cheese. Isolates with the same pulsed-field gel electrophoresis (PFGE)-pattern were detected on geographically distant dairies. This indicates that multiple sources and routes of contamination are important. To improve the safety of these products efforts to raise awareness of the importance of hygiene barriers and raw milk quality as well as improved process control can be suggested, e.g. use of starter cultures and monitoring of fermentation with a pH-meter. For future safety assessments, a better understanding of factors determining toxin production in these cheeses is needed.     

Short communication: Characterization of enterotoxin-producing Staphylococcus aureus isolated from mastitic cows

Staphylococcus aureus is not only a common cause of bovine mastitis, but also an agent of food poisoning in humans. In an attempt to determine whether staphylococci causing bovine mastitis could also cause food poisoning, 60 isolates of presumed S. aureus were isolated in the period between March and August 2017 from 3,384 routine, composite, quarter milk samples of individual cows raised on 12 dairy farms in central Italy. Seventeen out of 60 isolates were confirmed as S. aureus after coagulase, thermonuclease, and biochemical tests. These isolates were analyzed by PCR for the presence of the nuc, sea, seb, sec, sed, and see genes. The positive isolates were nuc, 100% (17); sea, 35.29% (6); seb, 5.88% (1); sec, 5.88% (1); sed, 29.41% (5); and see, 47.06% (8). The isolates were also tested with 2 enzyme immunoassay diagnostic kits, one for the screening detection of the production of staphylococcal enterotoxins (SEA, SEB, SEC, SED, SEE) and one for the detection of specific enterotoxin produced by each isolate. Seven out of 17 (41.18%) were enterotoxin producers: 7 produced SEA (41.18%), 1 SEB (5.88%), 1 SEC (5.88%), 5 SED (29.41%), and 6 SEE (35.29%). To further characterize the isolates, they were analyzed by the Kirby Bauer test for susceptibility to 13 antimicrobials (ampicillin, ciprofloxacin, kanamycin, tetracycline, gentamicin, methicillin, nalidixic acid, erythromycin, amoxicillin/clavulanic acid, streptomycin, vancomycin, neomycin, and enrofloxacin), and we detected resistance to ampicillin (52.94%), nalidixic acid (70.59%), erythromycin (5.88%), and amoxicillin/clavulanic acid (17.65%). The isolates were sensitive to the main classes of antimicrobials used for the treatment of bovine subclinical mastitis. The presence of enterotoxin-producing isolates of S. aureus in bovine milk means that a temperature abuse or a breakdown in the thermal treatment of the milk could present a food safety risk, particularly if all enterotoxigenic isolates could potentially produce SEA in milk.     

Prevalence,antimicrobial susceptibility,and molecular characterization of Staphylococcus aureus isolated from dairy herds in northern China

Staphylococcus aureus is one of the main pathogens involved in dairy cow mastitis. Monitoring of antibiotic use would prove useful to assess the risk of Staph. aureus in raw milk. The objective of this work was to investigate the prevalence of Staph. aureus strais isolated from raw milk in northern China, and to characterize antimicrobial susceptibility of these strains and their key virulence genes. In total, 195 raw milk samples were collected from 195 dairy farms located in 4 cities of northern China from May to September 2015. Out of 195 samples, 54 (27.7%) were positive for Staph. aureus. Among these 54 samples, 54 strains of Staph. aureus were isolated, and 16 strains were identified as methicillin-resistant Staph. aureus. The strains exhibited high percentages of resistance to penicillin G (85.2%), ampicillin (79.6%), and erythromycin (46.3%). Moreover, 72% of the strains showed resistance to more than one antimicrobial agent. Overall, 63% of penicillin-resistant strains possessed the blaZ gene, and 60% of the erythromycin-resistant strains possessed erm(A), erm(B), erm(C), msr(A), or msr(B) genes with 8 different gene patterns. All isolates resistant to gentamicin, kanamycin, and oxacillin carried the aac6'-aph2”, ant(4')-Ia, and mecA genes, respectively. Two tet(M)-positive isolates carried specific genes of the Tn916-Tn1545 transposon. The most predominant virulence genes were sec, sea, and pvl, which encode staphylococcal enterotoxins (sec and sea) and Panton-Valentine leukocidin, respectively. Thirty-two isolates (59.2%) harbored one or more virulence genes. The majority of Staph. aureus strains were multidrug resistant and carried multiple virulence genes, which may pose a risk to public health. Our data indicated that antimicrobial resistance of Staph. aureus was prevalent in dairy herds in northern China, and that antibiotics, especially penicillin G and ampicillin, to treat mastitis caused by Staph. aureus should be used with caution in northern China.     

Detection of genes for enterotoxins (ent) and toxic shock syndrome toxin-1 (tst) in mammary isolates of Staphylococcus aureus by polymerase-chain-reaction

During a two-year-period (1997–1998) 94 field strains of Staphylococcus aureus isolated from cases of bovine mastitis were investigated for the presence of genes for staphylococcal enterotoxin (ent) and toxic shock syndrome toxin-1 (TSST-1; tst). Polymerase-chain reaction (PCR) was performed using six pairs of relevant oligonucleotide primers. Thirty-four isolates were positive for one (18 strains) or two (16 strains) toxin genes. Three field strains were positive for enterotoxin A gene (sea), two for enterotoxin B gene (seb), 22 for enterotoxin C gene (sec), four for enterotoxin D gene (sed) and 19 for TSST1 gene (tst). The enterotoxin E gene (see) sequence was not found. The combination of sec and tst showed the highest incidence. A high correlation between PCR results and toxin protein detection by a commercial enzyme linked immunosorbent assay (ELISA) system and a reversed passive latex-agglutination (RPLA) test was observed. In these immunoassays, only one strain of S. aureus produced equivocal results for production of enterotoxin A, giving an overall concordance between genotypic and phenotypic identification of 98.9%.     

The Use of Multiplex PCR to Determine the Prevalence of Enterotoxigenic Staphylococcus aureus Isolated from Raw Milk,Feta Cheese,and Hand Swabs

Staphylococcus aureus (S. aureus) can cause mastitis in cattle and, therefore, can be present in milk. This study was undertaken to determine the prevalence of coagulase positive S. aureus and its enterotoxin genes sea, seb, and sec in isolates recovered from raw milk, feta cheese, and human hand swabs of milk and cheese handlers in Beni‐Suef province, Egypt. A total of 100 samples of raw milk and 50 samples of pasteurized‐milk feta cheese were collected. In addition, 50 hand swabs from milk handlers and 25 hand swabs from cheese handlers were examined for the presence of coagulase positive S. aureus. The isolates were characterized by multiplex PCR for detection of sea, seb, and sec genes, and for resistance to 5 classes of commonly used antibiotics. Twelve (12/100), 12 (6/50), and 17% (13/75) of milk, cheese, and hand swab samples, respectively, were positive for coagulase positive S. aureus. One isolate was obtained from each positive sample (31 isolates), and none contained genes for SEA or SEC production. Twenty‐five percent, 33%, and 31%, respectively, of the isolates contained the genes for SEB, resulting in 3%, 4%, and 5% of samples being positive for toxin producing coagulase positive S. aureus, respectively. At least one isolate was resistant to each of the antibiotics tested. Despite the low potential for SEB production shown, preventative measures, such as maintenance of the cold‐chain and good hygienic practices should be implemented to further reduce the potential risk to public health from SEB, and to reduce the spread of antimicrobial resistance.     

Genetic Diversity and Antibiotic Resistance Patterns of Staphylococcus Aureus Isolated from Leaf Vegetables in Korea

Staphylococcus aureus is an important foodborne pathogen on global basis. The current study investigated the genetic patterns in S. aureus isolates from leaf vegetables (n = 53). Additional isolates from livestock (n = 31) and humans (n = 27) were compared with the leaf vegetable isolates. Genes associated with toxins, antibiotic resistance, and pulsed‐field gel electrophoresis (PFGE) patterns were analyzed. At least 1 enterotoxin‐encoding gene (sea, seb, sec, sed, and see) was detected in 11 of 53 (20.75%) leaf vegetable isolates. When the agr (accessory gene regulator) grouping was analyzed, agr II was the major group, whereas agr IV was not present in leaf vegetable isolates. All S. aureus isolates from leaf vegetables were resistant to more than one of the antibiotics tested. Nineteen of 53 (35.85%) isolates from leaf vegetables exhibited multidrug‐resistance, and 11 of these were MRSA (methicillin‐resistant S. aureus). A dendrogram displaying the composite types of S. aureus isolates from 3 origins was generated based on the combination of the toxin genes, agr genes, antibiotic resistance, and PFGE patterns. The isolates could be clustered into 8 major composite types. The genetic patterns of S. aureus isolates from leaf vegetables and humans were similar, whereas those from livestock had unique patterns. This suggests some S. aureus isolates from leaf vegetables to be of human origin.     

Molecular characterization and antibiotic resistance of Staphylococcus spp. isolated from cheese processing plants

The aim of this research paper was to characterize coagulase-positive and coagulase-negative staphylococci from raw milk, Minas cheese, and production lines of Minas cheese processing. One hundred isolates from 3 different cheese producers were characterized using molecular approaches, such as PCR, molecular typing, and DNA sequencing. Staphylococcus aureus (88% of the isolates) was the most abundant followed by Staphylococcus epidermidis, Staphylococcus hyicus, and Staphylococcus warneri. Among the 22 enterotoxin genes tested, the most frequent was seh (62% of the isolates), followed by selx and ser. Hemolysin genes were widely distributed across isolates, and Panton-Valentine leukocidin and toxic shock syndrome toxin genes were also identified. Methicillin-resistant S. aureus were staphylococcal cassette chromosome mec III, IVa, IVd, and others nontypeable. In the phenotypic antibiotic resistance, multiresistant isolates were detected and resistance to penicillin was the most observed. Using spa typing, we identified several types and described a new one, t14969, isolated from cheese. These findings suggest that antibiotic resistance and potentially virulent strains from different sources can be found in the Brazilian dairy processing environment. Further research should be conducted with collaboration from regulatory agencies to develop programs of prevention of virulent and resistant strain dissemination in dairy products and the processing environment.     

Prevalence and characterization of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus isolated from retail yak butter in Tibet,China

This study aimed to investigate the prevalence, molecular characteristics and antibiotic resistance of Staphylococcus aureus isolates from yak butter in Tibet, China. A total of 218 yak butter samples were collected from retail stores in Tibet and screened for Staph. aureus. Furthermore, the virulence genes, resistance genes, antimicrobial susceptibility, and molecular typing [pulsed-field gel electrophoresis (PFGE), multilocus sequence typing, and staphylococcal protein A (spa) typing] of Staph. aureus isolates were detected. The results showed that 12.4% of yak butter samples were contaminated with Staph. aureus, including 5 samples positive for methicillin-resistant Staph. aureus (MRSA). Among all isolates, 96.3% harbored one or more virulence genes, including classical (sea and sec), novel enterotoxin-encoding genes (seh, sek, sel, and seq), and hemolysin genes (hla and hld). All isolates were resistant to at least 2 different antibiotic classes, and the isolates were most commonly resistant to sulfonamides, β-lactams, and erythromycin. For resistance genes, blaZ (74.1%) was most frequently detected, followed by dfrG (51.9%), erm(B) (22.2%), mecA (18.5%), tet(K) (14.8%), aph(2″)-Ia, aph(3′)-III, and ant(6)-Ia (11.1% for each), and erm(C) (7.4%). We detected 8 spa types, 6 sequence types (ST), and 5 clonal complex (CC) types. In addition, 1 isolate of Staph. aureus was nontypeable. We found that CC1-ST1-t559 (55.6%) was the most predominant clone, followed by CC59-ST59-t437 (11.1%), CC5-ST5-t002 (7.4%), CC1-ST1, CC1-ST1-t114, CC1-ST573-t4938, CC1-ST573-t8915, CC30-ST30-t021, and CC25-ST25-t167 (3.7% for each). For PFGE typing, a total of 5 clusters and 15 pulsotypes were generated, and some isolates from different samples showed indistinguishable pulsotypes. Our findings suggest that yak butter produced in Tibet, China, could be contaminated by Staph. aureus strains, including MRSA strains, carrying various virulence and resistance genes, representing multiple antimicrobial resistance phenotypes. The presence of potentially virulent and antibiotic-resistant Staph. aureus strains in yak butter poses a potential threat to consumers, and appropriate measures need to be taken in the production chain to reduce the occurrence of Staph. aureus in yak butter.     

Presence and growth prediction of Staphylococcus spp. and Staphylococcus aureus in Minas Frescal cheese,a soft fresh cheese produced in Brazil

Physical-chemical characteristics of Minas Frescal cheese (MFC) favor the growth of Staphylococcus spp. and allow the production of enterotoxins by specific strains. Here, we aimed to characterize the physical-chemical aspects (pH, storage temperature, and salt content) and the presence of Staphylococcus spp. in MFC samples (n = 50) to support a modeling study for the growth by this microorganism. Coagulase-positive staphylococci isolates were obtained and subjected to PCR assays to identify them as Staphylococcus aureus (nuc) and to detect staphylococcal enterotoxin-related genes (sea, seb, sec, sed, see). Staphylococcus aureus growth kinetics (maximum growth rate, Grmax, and lag time) were predicted based on ComBase model and MFC physical-chemical aspects. Mean counts of Staphylococcus spp. ranged from 3.3 to 6.7 log cfu/g, indicating poor hygiene practices during production. Selected isolates (n = 10) were identified as S. aureus, but none presented classical enterotoxin-related genes. pH, temperature, and salt content ranged from 5.80 to 6.62, 5°C to 12°C, and 0.85% to 1.70%, respectively. The Grmax values ranged from 0.012 to 0.419 log cfu/g per h. Independent of the storage temperature, the lowest Grmax values (0.012 to 0.372 log cfu/h) were obtained at pH 5.80 associated with salt content of 1.7%; independent of the pH and salt content, the best temperature to avoid staphylococcal growth was 7.5°C. Hygienic conditions during MFC production must be adopted to avoid staphylococcal contamination, and storage at temperatures lower than 7.5°C can prevent staphylococcal growth and the potential production of enterotoxins.     

Molecular characterization and clonal diversity of methicillin-susceptible Staphylococcus aureus in milk of cows with mastitis in Brazil

Mastitis is an important disease for the dairy industry worldwide, causing economic losses and reducing milk quality and production. Staphylococcus aureus is a worldwide agent of this intramammary infection, which also causes foodborne diseases. The objective of this study was to determine the frequency of methicillin-susceptible Staphylococcus aureus (MSSA) isolates in milk of mastitis cows in Brazil and to analyze the genetic lineages and the content of antimicrobial resistance genes and virulence factors among these isolates. Fifty-six MSSA isolates were recovered from 1,484 milk samples (positive for the California mastitis test) of 518 cows from 11 different farms in Brazil (representing 51% of total Staph. aureus obtained), and they were further characterized. Methicillin-susceptible Staphylococcus aureus were isolated from 3.7% of California mastitis test-positive tested milk samples and from 6.2% of tested mastitic cows. Methicillin-susceptible Staphylococcus aureus isolates were characterized by spa typing, agr typing, and multilocus sequence typing, and resistance and virulence traits were investigated by PCR. Seven spa types were identified among MSSA (% of isolates): t127 (44.6), t605 (37.5), t002, t1784, t2066 (1.8), and 2 new ones: t10856 (10.7) and t10852 (1.8). Five distinct sequence types (ST) were detected (% of isolates): ST1 (46.4), ST126 (37.5), ST133 (10.7), ST5 (3.6), and a novel ST registered as ST2493 (1.8). Resistances were detected for streptomycin, chloramphenicol, and tetracycline. One strain contained the chloramphenicol resistance gene (fexA; included within transposon Tn558) and 3 strains contained the tetracycline resistance gene [tet(K)]. Methicillin-susceptible Staphylococcus aureus strains were susceptible to most of the antibiotics studied and lacked the virulence genes of Panton-Valentine leukocidin (lukF/S-PV), toxic shock syndrome toxin 1 (tst), exfoliative toxin A (eta), and exfoliative toxin B (etb), as well as the genes of the immune evasion cluster. Methicillin-susceptible Staphylococcus aureus isolates were detected in a relatively low proportion of cows with mastitis (6.2%) and recovered isolates presented high diversity of genetic lineages, with CC1 and CC126 the predominant clonal complexes, and CC133 also being detected. Larger epidemiological studies with molecular characterization of isolates are required to deepen the knowledge on the circulating genetic lineages among the cow population with mastitis.     

Differentiation of toxigenic Staphylococcus aureusin staphylococcal isolates from prepared and frozen foods by combined arbitrarily primed polymerase chain reaction and DNA probe

In prepared and frozen flamenquín and hake fish fingers Staphylococcus aureus as sanitary hazards have been detected. In the present work, a combined method that includes an arbitrarily primed PCR (AP‐PCR) and a mixed DNA probe hybridisation designed for the enterotoxigenic genes sea, seb, sec, and sed will be assayed to differentiate enterotoxigenic S. aureus from other staphylococcal species isolated during the processing of prepared and frozen foods. From the protocols tested for the AP‐PCR, the highest number of amplification bands showing the best resolution was achieved at 30°C annealing and 35°C extension temperatures. Several staphylococci identified by a biochemical test as S. aureus showed in the AP‐PCR analysis different banding patterns to the references S. aureus. The isolates, were investigated by slot blot hybridisation for genes encoding A, B, C, and D staphylococcal enterotoxins to determine their enterotoxigenic potential. Several isolates characterised by the AP‐PCR analysis as S. aureus hybridised with the DNA probe mixture. The combined AP‐PCR and DNA probe hybridisation assayed was able to differentiate toxigenic S. aureus from other staphylococcal species from prepared and frozen foods. This method could be considered as microbial quality assurance in these products.